A systematic analysis of anti-diabetic medicinal plants from cells to clinical trials

Background Diabetes is one of the fastest-growing health emergencies of the 21st century, placing a severe economic burden on many countries. Current management approaches have improved diabetic care, but several limitations still exist, such as decreased efficacy, adverse effects, and the high cost of treatment, particularly for developing nations. There is, therefore, a need for more cost-effective therapies for diabetes management. The evidence-based application of phytochemicals from plants in the management of diseases is gaining traction. Methodology Various plants and plant parts have been investigated as antidiabetic agents. This review sought to collate and discuss published data on the cellular and molecular effects of medicinal plants and phytochemicals on insulin signaling pathways to better understand the current trend in using plant products in the management of diabetes. Furthermore, we explored available information on medicinal plants that consistently produced hypoglycemic effects from isolated cells to animal studies and clinical trials. Results There is substantial literature describing the effects of a range of plant extracts on insulin action and insulin signaling, revealing a depth in knowledge of molecular detail. Our exploration also reveals effective antidiabetic actions in animal studies, and clear translational potential evidenced by clinical trials. Conclusion We suggest that this area of research should be further exploited in the search for novel therapeutics for diabetes.


INTRODUCTION Statement of the problem
Diabetes mellitus is a metabolic disorder characterized by sustained hyperglycemia with numerous macrovascular and microvascular complications (Kharroubi, 2015;Stadlbauer et al., 2021;Zheng, Ley & Hu, 2018). Depending on the etiology, diabetes has been classified broadly into type 1 diabetes mellitus (T1DM), resulting from a deficiency in insulin production, and type 2 diabetes mellitus (T2DM), a defect in insulin action (Kharroubi, 2015). T1DM accounts for around 10% of cases, while T2DM accounts for about 90%. T2DM and its complications have contributed to a significant decrease in life expectancy (Zheng, Ley & Hu, 2018;Ogurtsova et al., 2017). The latest data suggests that 1 in 10 adults are living with diabetes, of which almost half are undiagnosed (Zheng, Ley & Hu, 2018), representing around 537 million citizens, and diabetes contributes to 1 in 9 deaths (Stadlbauer et al., 2021). These figures are projected to continue to rise. It is estimated that most countries devote 5-20% of healthcare expenditure to diabetes (Lin et al., 2020). The global spending to treat diabetes and its complications was US$760 billion in 2019, projected to increase to US$825 billion by 2030 (Stadlbauer et al., 2021;Modi, 2007). The disturbing increase in the prevalence of diabetes is a call for an augmented approach to the management of T2DM (Stadlbauer et al., 2021;Williams et al., 2020).
This review aims to collate, discuss, and present published data on the cellular and molecular effects of medicinal plants and phytochemicals on insulin signaling pathways to better understand the current trend in the use of plant products in the management of T2DM (Fig. 1). Furthermore, we have explored available information on the cell-biology of these medicinal plants that consistently produced hypoglycemic effects, with the intention of providing a reference point for the molecular basis of some of the more commonly used anti-diabetic plant extracts. We explored how these plant products might affect known insulin signaling systems and insulin effectors, and then extended our review into known effects on animal models and explored clinical trials of these compounds with the intention of providing a summary-view of related studies and a holistic overview of their use in rodent models or clinical trials. We conclude that plant products should be considered a vital tool in the armory for development of low-cost, effective anti-diabetic therapies.
The pathway from plant extract to effective therapy involves many steps. Plants, often identified from local knowledge/use, are a source of extract prepared using a range of approaches and the extracts screened using simple cell-based models such as Caco-2 cells or L6 skeletal myotubes for in vitro effects. Signaling pathways and effectors are used as surrogate assays for potential antidiabetic effects (e.g., glucose transport). Further work involves an examination of effects using rodent models of diabetes and clinical trials.

METHODOLOGY
We used a range of search terms to scan Google Scholar, PubMed, Science Direct, NIH National Library of Medicine and Scopus to retrieve published literature on medicinal plants and phytochemical effects on insulin signaling and effector pathways. Search terms focused on known signaling systems involved in propagating insulin signals (e.g., proteinqrynm_32 kinase-B/Akt (hereafter referred to as Akt); phosphoinositide-3 kinase (PI3K); glycogen synthase kinase-3 (GSK-3); AMP-activated protein kinase (AMPK); protein tyrosine phosphatase 1 B (PTP1B)); known effector molecules or processes (e.g., glucose transporters (GLUT) and GLUT4 storage vesicles (GSVs; also known as GSC-GLUT4 storage compartment); glucokinase (GCK)); glucagon secretion; lipolysis; lipogenesis; hepatic glucose output; and other molecules implicated in insulin action of insulin sensitivity, such as peroxisome proliferator-activated receptor gamma (PPARγ). Searches were performed between December 2021 and April 2022. We excluded articles not in English and not freely available via our institution (in this case the University of Strathclyde; <0.2% of articles retrieved) and no time limitation for publication date was employed. Our searches aimed to capture papers which described a potential effect on either signaling systems (e.g., PI3K, Akt etc.) or a biological output (e.g., glucose transport, GLUT4 mRNA). This was subsequently extended into whole animal studies and clinical trials.
Throughout we use the scientific and common names of the medicinal plants, and describe the chemistry used in the extraction process-a key consideration for studies of this type. In all tables, extracts are alphabetized by species unless multiple different species were used in the same study, in which case these are placed arbitrarily at the top of each table for clarity.

Effects of medicinal plants and phytochemicals on glucose transport and glucose transporters
Defective insulin-stimulated glucose transport is hallmark of T2DM (Ogurtsova et al., 2017;Nandabalan, Sujatha & Shanmuganathan, 2010;Drissi et al., 2021;Stadlbauer et al., 2016). Glucose transporters (GLUT) of the facilitative diffusion type are a multi-gene family of proteins which function to move glucose across cell membranes (Ogurtsova et al., 2017;World Health Organization, 2019;Singhal, Bangar & Naithani, 2012). Among the facilitative GLUT isoforms, GLUT4 is particularly important as it is expressed predominantly in skeletal and adipose tissues and accounts for post-prandial glucose disposal in these tissues (World Health Organization, 2019;Nandabalan, Sujatha & Shanmuganathan, 2010). Skeletal muscle contribute largely to a greater part of the total body mass in humans and it regulates several physiological processes including up to 85% of insulin-mediated glucose up-take through GLUT4 (Ahmad, Choi & Lee, 2020). Many studies have utilized this for therapeutic management of diabetes and in particular the role of extracellular matrix (Ahmad, Choi & Lee, 2020). Skeletal muscle contraction during exercise improves GLUT4 translocation to the cell membrane for glucose uptake and insulin-sensitivity (Jiang et al., 2013). Also, altered muscle glycogen synthesis play a major role in insulin resistance, and glycogen synthase, hexokinase, and GLUT4 are the major culprit involved in the skeletal muscle pathogenesis of type 2 dibetes (Petersen & Shulman, Figure 1 Schematic representation of antidiabetic development from plant to clinical trial. The pathway from plant extract to effective therapy involves many steps. Plants, often identified from local knowledge/use, are a source of extract prepared using a range of approaches and the extracts screened using simple cell-based models such as Caco-2 cells or L6 skeletal myotubes for in vitro effects. Signaling pathways and effectors are used as surrogate assays for potential antidiabetic effects (e.g., glucose transport). Further work involves an examination of effects using rodent models of diabetes and clinical trials.
Full-size  DOI: 10.7717/peerj.14639/ fig-1 2002; Saini, 2010). GLUT2 and GLUT5 are responsible for intestinal glucose and fructose uptake (Schreck & Melzig, 2021), while GLUT1 is present ubiquitously in all the body tissues (Galochkina et al., 2019). The dominant glucose transporters found in the small intestine are sodium-glucose linked transporter 1 (SGLT1) which accumulates glucose into adsorptive epithelial cells against its concentration gradient and GLUT2 which mediates movement of glucose from the epithelial cells into the blood (Schreck & Melzig, 2021); inhibition reduces the amount of glucose absorbed into the body. The hemodynamic activities of the glucose transporters have been extensively researched (Ogurtsova et al., 2017;World Health Organization, 2019;Singhal, Bangar & Naithani, 2012;Nandabalan, Sujatha & Shanmuganathan, 2010). Medicinal plants' products and phytochemicals that modify the action of the glucose transporters could significantly contribute to the search for effective drugs in the management of diabetes. Table 1 is a collation of studies of medicinal plants known to modulate glucose transport in cell lines. Some notable highlights of this extensive literature are discussed briefly below.
In one of the most comprehensive studies, Schreck & Melzig (2021) used Caco-2 cells exposed to a range of plant extracts to identify potential inhibitors of glucose transport. They reported between 40% to 80% reduction using the methanolic extracts of a range of plants including the fruits of Aronia melanocarpa, Valcheva-Kusmanova et al. (2007) Cornus officinalis, Crataegus pinnatifida, Lycium chinense, and Vaccinium myrtillus; the leaves of Brassica oleracea, Juglans regia, Peumus boldus, and the roots of Adenophora triphylla. The authors also reported 50% to 70% reduction by aqueous extract from the bark of Eucommia ulmoides and fruit skin of Malus domestica. These effects are likely acting via inhibition of GLUT1, the predominant transporter in these cells.
One of the key facets of insulin action is to drive the delivery of GLUT4 molecules from intracellular stores to the surface of fat and muscle cells, a process called 'translocation'. Stadlbauer et al. (2021) used CHO-K1 cells expressing GLUT4 and total internal reflection microscopy to identify GLUT4 translocation-inducing effects of some thirty plant extracts. Though the taxonomy of some of the plants were not fully defined, they included Hoodia, Sapindus mukorossi, Quillaja saponaria, Papaver, Castanea, Bitter orange (genus and species not specified), Oregon grape (genus and species not specified), Common daisy flowers, Rosebay willowherb leaves and Goldenrod flower as potential compounds that could be exploited as potential anti-hyperglycemic agents in the treatment of T2DM via effects on GLUT4 redistribution.
While the above study used a non-classical insulin target tissue (for good experimental reasons), others have focused upon more physiological cell systems. Through bioassay-guided fractionation, Kanaujia et al. (2010) reported two chebulinic acid derivatives from Capparis moonii with significant stimulatory effects on glucose uptake effects concomitant with increased IR-β, Insulin receptor substrate-1 (IRS-1) phosphorylation, and mRNA expression of GLUT4 and PI3K in L6 muscle cells. Carnosol from rosemary extract stimulated AMPK-dependent GLUT4 translocation with no effect on Akt phosphorylation in L6 myotubes (Vlavcheski et al., 2018). Methanolic extract of Gundelia tournefortii potentiated insulin-stimulated GLUT4 translocation to the plasma membrane in skeletal muscle L6 cells (Kadan et al., 2018). An aqueous extract of Cassia  abbreviata induced a two-fold increase in GLUT4 translocation in C 2 C 12 (mouse) skeletal muscle cells probably via activation of the canonical PI3K/Akt pathway (Kamatou, Ssemakalu & Shai, 2021). Naowaboot and colleagues reported the mechanism of antihyperglycemic effects of Morus alba leaf extract, including increasing glucose uptake via activation of the PI3K pathway and the plasma membrane translocation of GLUT4 in rat adipocytes (Naowaboot et al., 2012). Ethyl acetate extract and 3β-taraxerol isolated from Mangifera indica promoted increased GLUT4 translocation and glycogen synthesis in 3T3-L1 adipocytes (Nandabalan, Sujatha & Shanmuganathan, 2010). The study also noted the effect on glycogen synthesis was due to PI3K-dependent activation of Akt with subsequent inactivation of glycogen synthase kinase 3B (GSK3β) phosphorylation (discussed further below). The fruit of Citrullus colocynthis enhanced insulin-induced GLUT4 translocation and Akt phosphorylation in 3T3-L1 adipocytes (Drissi et al., 2021). Stadlbauer et al. (2016) screened further natural products as alternatives to insulin through quantitation of GLUT4 translocation in insulin-sensitive CHO-K1 and importantly extended their remarkable study into commercial adipocyte cells. Of the seven medicinal plants tested, Portulaca oleracea and Coccinia grandis were found to induce GLUT4 translocation together with increased glucose concentration uptake likely mediated by PI3K/Akt pathway in adipocytes (Stadlbauer et al., 2016).
Such findings, together with the many others noted in Table 1 and which space preclude detailed discussion of here, suggest natural products can drive re-distribution of GLUT4 to the plasma membrane in an insulin-mimetic manner. However, the effects are not confined to GLUT4. For example, Kang and colleagues reported upregulation of GLUT2 in the liver (and up-regulated GLUT4 in muscle) as the possible mechanism of the antidiabetic effects of Panax ginseng (black ginseng). Manzano & Williamson (2010) investigated the glucose uptake inhibition of polyphenols, phenolic acid, and tannins from strawberry (var. Abion) and apple (var. golden delicious) in Caco-2 intestinal cell monolayers and reported increased inhibition of GLUT2 and SGLT1 and reduced glucose intestinal bilayer transport. Enhancement of glucose uptake by mitragyna speciosa and mitragynine in rat L6 myotubes is associated with increased GLUT1 protein content (Purintrapiban et al., 2011). And the glucose uptake (GLUT4) enhancement activity of G. sylvestre in L6 myotubes alongside the amelioration of insulin resistance in the 3T3-L1 adipocytes cells have been reported (Kumar et al., 2016). One further study of note is the report that the methanol extract of the aerial part of Selaginella tamariscina enhanced glucose uptake in 3T3-L1 adipocytes, possibly by inhibition of PTP1B (El-Zein & Kreydiyyeh, 2011;Giro et al., 2009). Collectively, these studies reveal that medicinal plants have a range of action on glucose transport across multiple tissues of relevance to the treatment of diabetes.

Medicinal plants and phytochemical effects on PI3K and Akt activity
In the canonical insulin signaling pathway controlling glucose homeostasis, insulin receptor substrates are phosphorylated on tyrosine residues which then act as docking sites for downstream signaling molecules. Of particular note, IRS-1 recruits phosphoinositide 3kinase (PI3K) (Petersen & Shulman, 2018). PI3K phosphorylates phosphatidylinositol 4,5-biphosphate to form phosphatidyl-inositol 3,4,5-trisphosphate that in turn promotes Akt phosphorylation and activation (World Health Organization, Luna-Vital & De Mejia, 2018). Akt is an important nexus on the insulin signaling cascade because of its multi-substrate activities (Naowaboot et al., 2012;Nandabalan, Sujatha & Shanmuganathan, 2010;Tonks et al., 2013;Huang et al., 2018). Phosphorylation of Akt initiates a cascade of downstream events through many substrates, including phosphorylation of Akt substrate of 160 kDa (AS160), a RabGAP (GTPase activating protein); this in turn leads to GLUT4 translocation in muscle and adipose cells (Matschinsky, 2005;Sharma et al., 2021). Defects in Akt phosphorylation, as seen in impaired insulin activation, are associated with development of muscle and adipose insulin resistance in obesity and T2DM (Naowaboot et al., 2012;Joshi et al., 2013;Luna-Vital & De Mejia, 2018;Kim et al., 1999). The insulin PI3K/Akt pathway has been widely targeted in T2DM pharmacotherapy (World Health Organization, Naowaboot et al., 2012;Luna-Vital & De Mejia, 2018) and is important in the pathophysiology and therapy of other diseases (Nurcahyanti et al., 2021). Evidence of medicinal plants and phytochemicals modifying PI3K/Akt actions have been documented and are presented in Table 2. Some examples are briefly outlined below.
An ethyl acetate extract and 3β-taraxerol of Mangifera indica significantly activate GLUT4 translocation via a PI3K dependent pathway in 3T3-L1 adipocytes (Nandabalan, Sujatha & Shanmuganathan, 2010). Polysaccharides from corn silk (Maydis stigma) increased phosphorylation of Akt in a dose-dependent manner in L6 skeletal muscle myotubes (Guo et al., 2019). Similarly, phosphorylation of Akt in response to an extract of Folium sennae was described, together with a significant enhancement of GLUT4 translocation (Zhao et al., 2018a). Antidiabetic effect of alkaloids from the seeds of Nigella

Zhenjiang aromatic vinegar
Polyphenol-rich extract. PI3K Activation of PI3K/Akt pathway in IR-HepG2 cells. (Xia et al., 2021) glandulifera increased the activity of the PI3K/Akt pathway in L6 myotubes with a concomitant increase in glycogen synthesis and hexokinase activity (Tang et al., 2017). Four C21 steroidal glycosides A-D from G.sylvestre promoted GLUT4 translocation to the plasma membrane in L6 cells via activation of PI3K/AKT (Li et al., 2019a). These and other studies (Table 2) point to a significant number of useful PI3K/Akt modulators within medicinal plants.
Many studies have examined effects of medicinal plants in hepatoma cells, as liver is a key site of post-prandial glucose disposal with a notable emphasis on PI3K/Akt signaling ( Table 2). Heteropolysaccharides of Grifola frondosa (edible mushroom) increased IRS1/PI3K mRNA levels and enhanced insulin sensitivity . Polysaccharides of Dendrobium officinale increased the activity of PI3K and Akt and partially ameliorated symptoms in diabetic mice, pointing to a clear effect in a complex organism rather than simply in cell culture models . Inhibition of phosphorylated insulin receptor substrate-1(IRS-1), but activation of PI3K/Akt in insulin-resistant HepG2 cells by polyphenol-rich extract of Zhenjiang aromatic vinegar has been documented (Xia et al., 2021). Homogeneous polysaccharides from Sargassum pallidum ameliorate insulin resistance by upregulation of PI3K, Glycogen synthase (GS), and IRS-1 expression in insulin-resistant HepG2 cells (Cao et al., 2019). Monosaccharides from Anemarrhena asphodeloides Bunge exhibited hypoglycemic effects by activating PI3K/Akt, IRS-1signaling pathway, and inhibiting a-glucosidase activities in insulin-resistant-HepG2 cells (Chen et al., 2022). A natural flavonocoumarin (a-Methylartoflavonocoumarin) isolated from Juniperus chinensis was reported to activate PI3K/Akt pathway in insulin-resistant HepG2 cells (Jung et al., 2017). Thus, effects are evident both at the level of gene expression and activity mediated by a range of extracts.

Activity of medicinal plants and phytochemicals on glucokinase
Glucokinase (GCK) is important in the regulation of glucose metabolism in liver and pancreas. GCK is essential for pancreatic insulin secretion and hepatic insulin action via phosphorylation of glucose to glucose 6-phosphate (Naowaboot et al., 2012;Balakrishnan, Krishnasamy & Choi, 2018). Low GCK levels have been observed in T2DM (Haeusler et al., 2015) and could serve as a potential drug target for therapeutic intervention (Kim et al., 2013;Yang, Jang & Hwang, 2012). GCK is currently being targeted as therapeutic for T2DM (Matschinsky, 2009;Matschinsky & Wilson, 2019). Glucokinase activators have been advocated as an alternative approach to restoring and improving glycemic control in T2DM (Zhou et al., 2001;Towler & Hardie, 2007;Toulis et al., 2020). Several medicinal plants and phytochemicals with glucokinase activities have been recognized (Sharma et al., 2021). We have focused on the cellular and molecular glucokinase activities of medicinal plants in tissue culture models (Table 3).
Glucokinase activation by the leaf extract of Costus igneus (known in India as the 'insulin plant' for its purported anti-diabetic action) was examined in differentiated human hematopoietic stem cell (HSCs) as a model of β-cells. The extract increased GCK and inhibited of glucose-6-phosphatase activity by C. igneus, thereby improving glucose sensing, insulin production, and decreased gluconeogenesis (Kattaru et al., 2021). It was also reported that C. igneus profoundly increased insulin receptor and GLUT2 gene expression (Table 1)

Medicinal plants modifying activity of glycogen synthase kinase-3 (GSK-3)
GSK-3 inhibits glycogen synthase activity. Insulin phosphorylates GSK-3 and prevents glycogen synthase inactivation (Nabben & Neumann, 2016). This role of GSK-3 in the insulin signaling pathway provides a mechanistic approach to the use of GSK-3 inhibitors in the treatment of insulin-resistant diabetes. Two studies are worthy of comment. Ethanolic extract of Shilianhua (Sinocrassula indica Berge) was found to induce GSK-3β phosphorylation similarly to insulin in 3T3-L1 preadipocytes and rat skeletal L6 myoblasts, indicating a possible mechanism of antidiabetic activity (Yin et al., 2009). This extract also enhanced insulin-stimulated glucose consumption in L6 myotubes and H4IIE hepatocytes, and insulin-independent glucose uptake in 3T3-L1 adipocytes. The result also showed increased GLUT1 protein expression in 3T3-L1 and GLUT4 protein expression in L6 myotubes cells (Yin et al., 2009). Hot water reduction from the root of Sarcopoterium spinosum increased glycogen synthesis via induction of GSK-3 β phosphorylation in L6 myotubes (Sahuc, 2016). S. spinosum also enhanced basal insulin secretion in the pancreatic β-cells and inhibited isoproterenol-induced lipolysis in 3T3-L1 adipocytes (Sahuc, 2016).

Peroxisome proliferator-activated receptor-gamma (PPARγ) and medicinal plants
Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the nuclear receptor super-family which play integral roles in glucose and lipid metabolism (Mirza,  Althagafi & Shamshad, 2019). These receptors are targets for diabetes therapy and also for the treatment of cardiovascular disease, cancer, and inflammation (Mirza, Althagafi & Shamshad, 2019). We present the effects of various medicinal plants on PPARγ activity and gene expression (Table 4). Chloroform extract of the fruit of Momordica charantia has been reported to significantly increase PPARγ gene expression 2.8-fold, comparable to the insulin sensitizer rosiglitazone (2.4-fold) in L6 myotube skeletal muscle cells (Kumar et al., 2009). Huang et al. (2005 demonstrated that Punica granatum flower extract and ethyl acetate fractions enhanced PPARγ gene expression and protein levels in a macrophage cell line. Increased mRNA of PPARγ (and GLUT4) by Nymphaea nouchali seed extract in 3T3-L1 adipocytes as the possible mechanism of its anti-hyperglycemic effect was reported (Parimala et al., 2015). Exposure of 3T3-L1 adipocytes to an ethanol extract of Miconia increased mRNA of PPARγ by 1.4-fold and inhibited a-amylase and a-glucosidase. The extract also increased lipid accumulation by around 30% as a possible anti-diabetic mechanism of action (Ortíz-Martinez et al., 2016). Upregulation of PPARγ together with GLUT4, SREBP and FAS expression was observed in 3T3-L1 adipocytes treated with the leaf extract of Moringa concanensis (Balakrishnan, Krishnasamy & Choi, 2018). Effects in muscle models have also been reported: Kim et al. (2013) reported increased transcription activity and mRNA levels of PPARγ in C 2 C 12 myotubes by Boehmeria nivea ethanol leaf extract and Korean red peppers (Yeongyang korea) increased glucose uptake in C 2 C 12 via increased transcriptional activity of PPARγ (Yang, Jang & Hwang, 2012).

AMP-activated protein kinase (AMPK)
AMP-activated protein kinase (AMPK) is a known energy sensor for metabolic homeostasis (Steinberg & Carling, 2019) which plays a central role in regulating lipid and protein metabolism together with fatty acid oxidation and muscle glucose uptake (Sathishsekar & Subramanian, 2005). AMPK plays a crucial role in insulin sensitivity, which explains its place as a potential drug candidate for T2DM therapy (Tasic et al., 2021;Hawkins et al., 2021). AMPK systems have been said to be partly responsible for the health benefits of exercise and AMPK is an important downstream effector of metformin. It has also been proposed as a possible target for novel drugs in managing obesity, type 2 diabetes, and metabolic syndrome (Hu, Zeng & Tomlinson, 2014;Hosseini et al., 2014;Kim et al., 2016). Ethnopharmacological investigators have reported that several medicinal plants modulate the activity of AMPK in cell models, and we have summarized these reports in Table 5, and highlight a few notable studies below. Ethanolic extract and phytochemical compounds from Cimicifuga racemosa mediate increased AMPK activity in fully differentiated HepaRG cells and is a possible mechanism of antidiabetic activity (Moser et al., 2014). Yuan & Piao (2011) reported activation of AMPK by the petroleum ether fraction of Artemisia sacrorum in HepG2 cells. They showed increased phosphorylation of AMPK (on T172), acetyl-CoA carboxylase (ACC; reside S79), and GSK-3β and reported concomitant downregulation of phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase). Similarly, Zheng et al. (2016) exploring the anti T2DM activity of Entada phaseoloides in primary mouse hepatocytes and HepG2 cells, reported suppression of hepatic gluconeogenesis via activation of the AMPK signaling pathway and Akt/GSK-3β. Extract of Rosmarinus officinalis significantly increased glucose consumption in HepG2 cells via increased phosphorylation of AMPK and ACC and potentially increased liver glycolysis and fatty acid oxidation (Tu et al., 2013). Thus, numerous examples of medicinal plants exerting effects via AMPK in hepatoma cell lines have been described (Table 5).
Effects mediated via AMPK have been reported in other cell types. These include an alcoholic extract of Artemisia dracunculus enhanced insulin release from β-cells isolated from mouse and human islets via activation of AMPK and suppressed LPS/IFNγ-induced inflammation. Effects on glucose transport in muscle lines include an extract of Crocus sativus (saffron) which increased glucose uptake and insulin sensitivity in C 2 C 12 myotubes by increased phosphorylation of AMPK in a dose and time-dependent manner (Kang et al., 2012) and compounds isolated from the seed of Iris sanguinea was reported to be via AMPK and ACC phosphorylation in the same cell type .
Ethanolic extract and isolated compound (β-sitosterol) from Malva verticillata seed significantly increased activation of AMPK as the molecular mechanism for glucose uptake in L6 myotubes (Jeong & Song, 2011). Triterpenoids from the stem of Momordica charantia have been reported to overcome insulin resistance in FL83B and C2C12 via AMPK activation (Cheng et al., 2008). Hence, the effects of such compounds on AMPK is an active and vigorous area of research.

Studies in animal models
The process of drug development encompasses pre-clinical experimentation (in vitro, in silico, and in vivo) leading ultimately to clinical trials in humans. To understand if there is ongoing vertical research towards developing antidiabetic agents from these medicinal plants, we reviewed their exploitation in experimental animal models as summarized in Table 6. Some highlights are discussed below.
Aronia melanocarpa fruit juice was found to mediate a dose-dependent decrease in plasma glucose and triglyceride levels in streptozotocin-induced hyperglycemic rats (Lee et al., 2016;Mazibuko et al., 2013), corresponding to the observations on glucose transport in Caco-2 cells alluded to above (Table 1) (Schreck & Melzig, 2021). Similarly, the antidiabetic and hyperlipidemic effects of Crataegus pinnatifida were investigated in high fat-fed mice. The results showed decreased glucose production and triglyceride synthesis via induction of AMPK phosphorylation (Shih et al., 2013), compared with inhibition of SGLT1 and GLUT2 in Caco-2 cells (Schreck & Melzig, 2021). These provide a good example of studies in cell lines being translated into animal models.
Regulate glucose and lipid metabolism through activation of AMPK and PPAR pathways in HepG2 cells.

Vigna angularis
Hot water extract and polysaccharides from the leaves.

Mice and Rats
Reduced FBG, an triglycerides, but increased HDL-C, and reduction in diabetes progression. (Zheng et al., 2011;Xu et al., 2018;Itoh et al., 2009)  The fruit of Vaccinium myrtillus was reported to significantly reduce serum glucose, total cholesterol, low density lipoprotein cholesterol, and very low density lipoprotein cholesterol, and triglycerides in alloxan-induced hyperglycemic adult male Wistar rats (Asgary et al., 2016).
Juglans regia extracts reduce blood glucose levels in diabetic mice (Kamyab et al., 2010), ameliorated streptozotocin-induced diabetic peripheral neuropathy in rats (Nasiry et al., 2017), and significantly decreased blood glucose, glycosylated hemoglobin, LDL, triglycerides, and total cholesterol in Wistar rats (Mohammadi et al., 2011). An aqueous extract of the seeds of Momordica charantia reduced blood glucose level, glycosylated hemoglobin, lactate dehydrogenase, glucose-6-phosphatase, fructose-1,6-biphosphatase, and glycogen phosphorylase but increased the activities of glycogen synthase and hexokinase in streptozotocin-induced diabetic rats, providing clear evidence of a systematic and programmed action on key metabolic activities (Sathishsekar & Subramanian, 2005). Similarly, polysaccharides of Dendrobium officinale reduced blood glucose level, glycated serum protein, total cholesterol, LDL-C, and increased HDL-C in type 2 diabetic rats (Chen et al., 2020). Cimicifuga racemosa extracts from rhizomes and roots reduced body weight, plasma glucose, improved glucose metabolism, and increased insulin sensitivity in obese diabetic mice (Moser et al., 2014). Tarralin TM , an ethanolic extract of Artemisia dracunculus, significantly lowered blood glucose concentrations and PEPCK in diabetic KK-A y mice (Ribnicky et al., 2006). Li et al. (2019a) described the effects of Gymnemic acid isolated from G. sylvestre on insulin signalling pathways in the type 2 diabetic rats as activation of PI3K/AKT together with AMPK phosphorylation. Such studies exemplify the power of medicinal plants in the amelioration of metabolic disturbances, and Table 5 summarizes the wide array of studies relevant to diabetes research.

Medicinal plants in clinical trials
The process of drug discovery necessitates that a drug molecule or product that has successively passed through the preclinical stage of drug development is carefully tested in clinical trials. We reviewed those plants that progressed to clinical trials and present our findings in Table 7. As this area is particularly important, we have provided some detail of key studies in the sections below.
Prospective open-label clinical trials of Alixir 400 PROTECTÒ (standardized extract of Aronia melanocarpa) in 143 patients demonstrated controlled glycemia, blood pressure improvement, and beneficial effects on LDL-C, triglycerides and total cholesterol, and was of significant (p < 0.05) overall benefit in diabetic hypertensive patients (Tasic et al., 2021). Similarly, a meta-analysis of controlled clinical trials carried out on Aronia melanocarpa daily supplementation revealed significant (p < 0.05) decreases in total cholesterol, blood pressure and a reduction in cardiovascular and diabetic risk factors, clearly supporting a useful role in therapy (Hawkins et al., 2021).
Lipid lowering effects are also a common feature of clinical trials with medicinal plants. Hu, Zeng & Tomlinson (2014) demonstrated the beneficial effects of a multi-herb formula containing Crataegus pinnatifida for dyslipidemia in a randomized double-blind, placebo-controlled trial, reporting decreased plasma lipids, glucose levels, HbA1c, and LDL-C at 95% CI. A randomized, double-blind placebo-controlled trial of Juglans regia leaf extract resulted in a significant decrease (p < 0.05) in fasting blood glucose levels, triglycerides, total cholesterol and HbA1c compared with placebo (Hosseini et al., 2014). Similar beneficial effects were reported in further trials (Rabiei et al., 2018) including beneficial effects in patients with coronary artery disease with significant decreases (p = 0.04) in total cholesterol, BMI, and LDL (Hajizadeh-Sharafabad et al., 2016). A clinical investigation of oral administration of Portulaca oleracea seeds in 30 T2DM subjects revealed a significant decrease (p < 0.001) in serum levels of triglycerides, total cholesterol, LDL-C, and liver enzymes, but increased (p > 0.001) levels of HDL-C and albumin (El-Sayed, 2011). Purslane (Portulaca oleracea capsule) also drove a significant difference (p > 0.01) in the triglycerides, liver enzymesand fasting blood glucose in 74 people with T2DM in a randomized double-blind, placebo-controlled clinical trial. An improvement in both insulin resistance, and LDL-C levels was also reported (Darvish Damavandi et al., 2021).
Decreased blood glucose levels are often used as a key outcome. A 14-day clinical investigation involving 26 people with T2DM on a low (0.5 g/kg) and high (1 g/kg) doses of aqueous and alcoholic extract of the powdered leaves of Mangifera indica showed a significant decrease in blood glucose levels in all groups (Waheed, Miana & Ahmad, 2006). Similarly, both raw and processed seeds of Ocimum basilicum in patients with diabetes and dyslipidemia revealed beneficial effects including decreased blood glucose, a reduction in body mass index, triglycerides, LDL-C, and decreased HDL-C at 5% and 1% levels of significant (Arivuchudar, Nazni & Uvaraj, 2022). A significant decrease (p < 0.05) in the fasting blood glucose and HbA1c levels were observed in people with T2DM after 2 month treatment with the fruit capsule of Citrullus colocynthis (Jang et al., 2008).
In a randomized, double-blind, crossover study of 60 diabetic subjects receiving Salacia oblonga extract, Williams et al. (2007) reported significant decrease (p < 0.05) in glyceamia and insulinemia in patients after high carbohydrate meal. A 4-week randomized doubleblind, placebo-controlled clinical trial of fermented red ginseng (Panax ginseng) involving 42 patients with impaired fasting glucose or T2DM also showed significant decrease (p < 0.01) in postprandial glucose levels and increased postprandial insulin levels compared to the placebo group (Oh et al., 2014). A further study supported these conclusions (Vuksan et al., 2008).
Many other studies, highlighted in Table 7, have shown pronounced and beneficial effects.
Not all data are conclusive. The results of a 60-day randomized-placebo clinical trial by Hasanzade and colleagues using Cinnamomum cassia in 70 people with T2D revealed no significant difference (p > 0.05) between the test and placebo (Hasanzade et al., 2013). On the other hand, Hoehn and Stockert in a smaller trial reported significant decrease in blood sugar levels of the patients taking 1,000 mg Cinnamomum cassia capsule for 12 weeks (Hoehn & Stockert, 2012). An aqueous extract of G. sylvestre (GS4) 400 mg/day used over 18 to 20 months supplementation drove significant decreases (p < 0.001) in blood glucose, glycosylated haemoglobin and glycosylated plasma protein in 22 patients (Baskaran et al., 1990). It should also be clearly noted that many of the clinical studies performed to date include relatively small numbers of patients. Larger studies will provide impetus for more work in this area.

CONCLUSION AND FUTURE PERSPECTIVES
Diabetes mellitus-related morbidity and mortality continues to increase globally and necessitates urgent action to identify and drive novel therapies which can be widely used in under-developed economies. Our review reveals that antidiabetic drugs of herbal origin can play a modulatory role in insulin signaling pathways and drive metabolically relevant changes in insulin action, such as elevated glucose transport. Tissue culture systems have provided key insight into the molecular mechanisms of the phytochemicals beneficial to diabetic patients and have contributed both mechanistic insight and facilitated the development of more clinically-facing treatments. Among the plants we reviewed in tissue culture systems, close to half (45%) have been investigated for their antidiabetic activities in mammals (rats, mice, and rabbits) and 4% have been tested in human clinical trials. The positive outcomes reported in these clinical trials should be recognized as providing a new impetus to phytobiology research as an effective treatment for insulin resistance and diabetes. In future, larger-scale clinical trials are clearly warranted given the largely positive effects of many of these natural products. There is a need to screen larger numbers and citizens of different genetic backgrounds to identify potential population-specific benefits. Similarly, the coupling of phytochemical studies to genomic data may offer a powerful means to develop combination therapies and more personalized medicine approaches. Omale and Gwyn Gould (AREF-308-OMALE-F-C0818) and a grant from Diabetes UK (to Gwyn W Gould; 18/0005847). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.